Medicine for treating cancer by administering a toll-like receptor agonist and LAG-3 IgG fusion protein

ABSTRACT

The present invention provides a medicine comprising a Toll-like receptor agonist, LAG-3 protein, a variant or derivative thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.16/341,415, which is the U.S. National Stage application ofPCT/JP2017/015227, filed Apr. 14, 2017, which claims priority fromJapanese application no. JP 2016-200227, filed Oct. 11, 2016.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 22, 2022, isnamed sequence.txt and is 6,658 bytes.

TECHNICAL FIELD

The present invention relates to a medicine or the like comprising aToll-like receptor agonist and LAG-3 protein, a variant or derivativethereof.

BACKGROUND ART

Recently, cancer immunotherapies targeting cancer antigens expressedspecifically in cancer cells have been developed. Of them, a “cancervaccine therapy” is a method of inducing regression of cancer byadministering a cancer antigen directly to patients to induce an immuneresponse specific to a cancer antigen in the patient's body. As thecancer antigen to be administered to a patient, e.g., a cancer-antigenprotein itself, a cancer-antigen derived peptide, a nucleic acidencoding them, a dendritic cell presenting a cancer antigen and a cancercell itself, are used.

To enhance induction of an immune response by a cancer antigen, anadjuvant is administered together with the cancer antigen. As theadjuvant, e.g., cytokines stimulating various immunocompetent cells andToll-like receptor (TLR) agonists are used.

As the Toll-like receptor agonist (TLR agonist) serving as an adjuvant,any one of TLR1 to TLR10 agonists can be used (for example, Non PatentLiteratures 1 and 2). For example, TLR3, which recognizes virus-deriveddouble-stranded RNA and accelerates production of type I interferonexerting a strong antiviral action, is a molecule of the innate immunesystem. A double-stranded RNA analogue, Poly I:C (also called asPolyinosinic:polycytidylic acid), which is known as a TLR3 agonist, isknown to be used as a vaccine adjuvant (for example, Patent Literature1). Also, TLR9, which recognizes a bacterium- or virus-derivedunmethylated CpG DNA and exerts an action, is a molecule of the innateimmune system. CpG ODN (synthetic nucleic acid CpG oligodeoxynucleotide)serving as a TLR9-ligand is known to have an adjuvant effect forvaccine.

It is known that LAG-3 is also used as an adjuvant for vaccine (forexample, Patent Literature 2). LAG-3 is a posttranslational product oflymphocyte activation gene 3 and also called as CD223. LAG-3 binds to aMHC Class II molecule to negatively control proliferation of activated Tcells and homeostasis maintenance of T cells, plays an important role inregulatory T cell (Treg) function and is also known to be involved inhomeostatic regulation of plasmacytoid dendritic cells.

CITATION LIST Patent Literature

-   Patent Literature 1: National Publication of International Patent    Application No. 2011-506309-   Patent Literature 2: National Publication of International Patent    Application No. 2001-510806

Non Patent Literature

-   Non Patent Literature 1: Oncolmmunology 1: 5, 699-716; August 2012-   Non Patent Literature 2: Oncolmmunology 2: 8, e25238; August 2013

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a medicine comprising anovel combination of adjuvants.

Solution to Problem

The present inventors conducted various studies using various adjuvantssingly or in combination. As a result, they found a novel combination ofadjuvants serving as a medicine useful for, for example, inducing cancerimmunity.

More specifically, the present invention is as follows.

[1]

A medicine comprising

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof.

[2]

The medicine according to [1] for combined administration of

the Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof.

[3]

The medicine according to [1] or [2], wherein the Toll-like receptoragonist is Toll-like receptor 3 agonist or a Toll-like receptor 9agonist.

[4]

The medicine according to any one of [1] to [3], in which the Toll-likereceptor agonist is Poly I:C or a salt thereof.

[5]

The medicine according to [4], wherein the Poly I: C is Poly ICLC.

[6]

The medicine according to any one of [1] to [5], wherein the LAG-3protein, a variant or derivative thereof is a fusion protein of LAG-3protein and IgG.

[6′]

The medicine according to any one of [1] to [6], further comprising asubstance for inducing a specific immune response to at least one cancercell.

[6″]

The medicine according to [6′], for combined administration of

the Toll-like receptor agonist,

LAG-3 protein, a variant or derivative thereof, and

the substance for inducing a specific immune response to at least onecancer cell.

[7]

The medicine according to any one of [1] to [6], further comprising atleast one cancer-antigen derived peptide.

[8]

The medicine according to [7], wherein the at least one cancer-antigenderived peptide is an HSP70-derived peptide or a GPC3-derived peptide.

[9]

The medicine according to [7] or [8], comprising at least onecancer-antigen derived peptide selected from the group consisting of anHSP70-derived peptide and a GPC3-derived peptide,

Poly ICLC and

LAG-3 protein or a fusion protein of LAG-3 protein and IgG.

[10]

The medicine according to [9], comprising an HSP70-derived peptidehaving an amino acid sequence represented by SEQ. ID No: 7 or aGPC3-derived peptide having an amino acid sequence represented by SEQ.ID No: 16.

[11]

The medicine according to any one of [7] to [10], comprising at leasttwo types of cancer-antigen derived peptides.

[12]

The medicine according to [11], comprising an HSP70-derived peptide anda cancer-antigen derived peptide except an HSP70-derived peptide.

[13]

The medicine according to [11], comprising a GPC3-derived peptide and acancer-antigen derived peptide except a GPC3-derived peptide.

[14]

The medicine according to [11], comprising an HSP70-derived peptide anda GPC3-derived peptide.

[15]

The medicine according to [14], comprising an HSP70-derived peptidehaving an amino acid sequence represented by SEQ. ID No: 7 and aGPC3-derived peptide having an amino acid sequence represented by SEQ.ID No: 16.

[16]

The medicine according to [12], comprising an HSP70-derived peptide, acancer-antigen derived peptide except an HSP70-derived peptide,

Poly ICLC and

LAG-3 protein or a fusion protein of LAG-3 protein and IgG.

[17]

The medicine according to [13], comprising

a GPC3-derived peptide, a cancer-antigen derived peptide except aGPC3-derived peptide,

Poly ICLC and

a LAG-3 protein or a fusion protein of LAG-3 protein and IgG.

[18]

The medicine according to [14], comprising

an HSP70-derived peptide, a GPC3-derived peptide,

Poly ICLC and

LAG-3 protein or a fusion protein of LAG-3 protein and IgG.

[19]

The medicine according to [18], comprising

an HSP70-derived peptide having an amino acid sequence represented bySEQ. ID No: 7 and a GPC3-derived peptide having an amino acid sequencerepresented by SEQ. ID No: 16.

[20]

The medicine according to any one of [1] to [19], for use in a cancervaccine therapy.

[21]

The medicine according to any one of [1] to [20], wherein the medicineis an anti-cancer agent.

[22]

An adjuvant comprising

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof, for use in inducing aspecific immune response to a cancer cell or for use in a cancer vaccinetherapy.

[23]

A combination comprising

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof, for use in inducing aspecific immune response to a cancer cell or in a cancer vaccinetherapy.

[24]

A method for treating or preventing a disease in a patient, comprisingadministering

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof, to a patient in needthereof.

[25]

A method for inducing a specific immune response to a cancer cell,comprising administering

a Toll-like receptor agonist and

LAG-3 protein, a variant or derivative thereof, to a patient in needthereof.

Advantageous Effects of Invention

According to the present invention, it is possible to provide a medicinecomprising a new combination of adjuvants.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic protocol of an experiment for comparing effectsof adjuvants in a cancer vaccine using a cancer-antigen derived peptide.

FIG. 2 is a graph showing changes in tumor size of Group 1, in which PBSwas used as a control in the experiment of FIG. 1 .

FIG. 3 is a graph showing changes in tumor size in Group 2, in which IFAwas used as an adjuvant in the experiment of FIG. 1 .

FIG. 4 is a graph showing changes in tumor size in Group 3, in which PTwas used as an adjuvant in the experiment of FIG. 1 .

FIG. 5 is a graph showing changes in tumor size in Group 4, in whichPoly I:C was used as an adjuvant in the experiment of FIG. 1 .

FIG. 6 is a graph showing changes in tumor size in Group 5, in which acombination of Poly I:C and CpG was used as an adjuvant in theexperiment of FIG. 1 .

FIG. 7 is a graph showing change in tumor size in Group 6, in whichLAG-3Ig was used as an adjuvant in the experiment of FIG. 1 .

FIG. 8 is a graph showing changes in tumor size in Group 7, in which acombination of LAG-3Ig and Poly I:C was used as an adjuvant in theexperiment of FIG. 1 .

FIG. 9A shows images of a tumor tissue stained with hematoxylin-eosin inthe same experiment as in FIG. 1 .

FIG. 9B shows images of cell nuclei and immune cells of a tumor tissuestained with a fluorescent dye in the same experiment as in FIG. 1 .

FIG. 10 is a graph showing the measurement results (increase) of tumorsize after tumor cells were again inoculated to a mouse in which anincrease in tumor size was once successfully suppressed by a cancervaccine (LAG-3Ig and Poly I:C were used as adjuvants) in the sameexperiment as in FIG. 1 .

FIG. 11A is a graph showing the measurement results of proliferativeability of immune cells, which were prepared by taking a lymph node froma mouse inoculated with a cancer vaccine in the same experiment as inFIG. 1 , separating the immune cells from the lymph node, and which wereco-culturing with tumor cells previously inactivated by radiationexposure.

FIG. 11B shows graphs showing the measurement results of cytokines inthe cell supernatant of the same co-culture as in the experiment in FIG.11A.

FIG. 12A shows the measurement results of expression of cell surfacemarker molecules, PD-1, BTLA, TIGIT and LAG-3, present on the surface ofCD8 positive immune cells, which were prepared by taking a lymph nodefrom a mouse inoculated with a cancer vaccine in the same experiment asin FIG. 1 and separating the immune cells from the lymph node.

FIG. 12B shows the measurement results of expression of cell surfacemarker molecules, PD-1, BTLA, TIGIT and LAG-3, present on the surface ofCD4 positive immune cells, which were prepared by taking a lymph nodefrom a mouse inoculated with a cancer vaccine in the same experiment asin FIG. 1 and separating the immune cells from the lymph node.

FIG. 13A is a graph showing measurement results of proliferative abilityof immune cells, and FIG. 13B is a graph showing measurement results ofIFN-γ production.

FIG. 14 is a graph showing changes in tumor size of Group 1, in whichPBS was used as a control.

FIG. 15 is a graph showing changes in tumor size of Group 2, in whichPoly I:C was used as an adjuvant.

FIG. 16 is a graph showing changes in tumor size in Group 3, in which acombination of LAG-3Ig and Poly I:C was used as an adjuvant.

FIG. 17 is a graph showing changes in tumor size in Group 4, in which acombination of LAG-3Ig and Poly I:C, alone was used without using PIApeptide.

FIG. 18 is a graph showing changes in tumor size of Group 5, in whichRIBOXXOL was used as an adjuvant.

FIG. 19 is a graph showing changes in tumor size of Group 6, in which acombination of LAG-3Ig and RIBOXXOL was used as an adjuvant.

FIG. 20 is a graph showing changes in tumor size of Group 7, in which acombination of LAG-3Ig and MPL was used as an adjuvant.

FIG. 21 is a graph showing changes in tumor size of Group 8, in which acombination of LAG-3Ig and Imiquimod was used as an adjuvant.

FIG. 22 is a graph showing changes in tumor size of Group 9, in which acombination of LAG-3Ig and CpG was used as an adjuvant.

FIG. 23 is a graph showing changes in tumor size of Group 1, in whichIFA was used as an adjuvant.

FIG. 24 is a graph showing changes in tumor size of Group 2, in whichPoly I:C was used as an adjuvant.

FIG. 25 is a graph showing changes in tumor size of Group 3, in whichLAG-3Ig was used as an adjuvant.

FIG. 26 is a graph showing changes in tumor size in Group 4, in which acombination of LAG-3Ig and Poly I:C was used as an adjuvant.

FIG. 27A is a graph showing IFN-γ productions from immune cells isolatedfrom patient's blood and cultured after an HSP70-derived peptide, aGPC3-derived peptide and a combination of Poly ICLC (product name:“Hiltonol”) and LAG-3Ig (IMP321) were administered 10 times toesophageal cancer patients.

FIG. 27B is a graph showing IFN-γ productions from immune cells isolatedfrom patient's blood and cultured after an HSP70-derived peptide, aGPC3-derived peptide and a combination of Poly ICLC (product name:“Hiltonol”) and LAG-3Ig (IMP321) were administered 10 times to livercancer patients.

FIG. 28A is a graph showing IFN-γ productions from immune cells isolatedfrom patient's blood and cultured after an HSP70-derived peptide, aGPC3-derived peptide and a combination of Poly ICLC (product name:“Hiltonol”) and LAG-3Ig (IMP321) were administered 4 times to livercancer patients.

FIG. 28B is a graph showing IFN-γ productions from immune cells isolatedfrom patient's blood and cultured after an HSP70-derived peptide, aGPC3-derived peptide and a combination of Poly ICLC (product name:“Hiltonol”) and LAG-3Ig (IMP321) were administered 8 times to rectalcancer patients.

FIG. 28C is a graph showing IFN-γ productions from immune cells isolatedfrom patient's blood and cultured after an HSP70-derived peptide, aGPC3-derived peptide and a combination of Poly ICLC (product name:“Hiltonol”) and LAG-3Ig (IMP321) were administered 8 times to esophagealcancer patients.

DESCRIPTION OF EMBODIMENTS

The present invention will be more specifically described below by wayof embodiments; however, the present invention is not limited to theembodiments and can be modified in various ways and carried out.

A medicine according to the present invention includes a Toll-likereceptor agonist (TLR agonist) and LAG-3 protein, a variant orderivative thereof.

In the specification, the “Toll-like receptor agonist (TLR agonist)”refers to a molecule, which binds to any one of Toll-like receptors andprovides the same stimulus as the stimulus provided by a natural ligandbound to TLR. As the TLR agonist, TLR agonists described in Non PatentLiteratures 1 and 2, Trends in Immunology, Vol. 30, No. 1, 23-32,Immunity 33, Oct. 29, 2010, 492-503, World Journal of Vaccines, 2011, 1,33-78 and Oncolmmunology 1:6, 894-907; September 2012, are known. TheseTLRs known in the art each can be used as the TLR agonist in the presentinvention.

As the TLR agonist, TLR1 to TLR10 agonists are mentioned, for example, aTLR3 agonist, TLR4 agonist, TLR7 agonist, TLR8 agonist, TLR9 agonist orTLR10 agonist can be used. Of them, a TLR3 agonist or a TLR9 agonist maybe used as the TLR agonist.

In the specification, the “TLR1 agonist” refers to a molecule whichbinds to TLR1 and provides the same stimulus as the stimulus provided bya natural ligand bound to TLR1. TLR1 recognizes e.g., a lipoprotein, toactivate the innate immune system.

Examples of the TLR1 agonist include a lipoprotein, Triacylatedlipopeptides and Zymosan.

In the specification, the “TLR2 agonist” refers to a molecule whichbinds to TLR2 and provides the same stimulus as the stimulus provided bya natural ligand bound to TLR2. TLR2 recognizes, e.g., Acylatedlipopeptides, to activate the innate immune system.

Examples of the TLR2 agonist include SMP-105, Acylated lipopeptides,Amphotericin B, Atypical LPS, Byglican, Forins, Glyco(phospho)lipids,Lipoteichoic acid, Peptidoglycan, Phenol soluble modulin and Zymosan.

In the specification, the “TLR3 agonist” refers to a molecule whichbinds to TLR3 and provides the same stimulus as the stimulus provided bya natural ligand bound to TLR3. TLR3 recognizes a virus-derived doublestranded RNA to activate the innate immune system. As the TLR3 agonist,a synthetic double stranded polynucleotide, Poly I:C, having ananalogous structure to double stranded RNA is known; however, the TLR3agonist is not limited to this.

A salt of Poly I:C may be used in the present invention; for example, asodium salt thereof can be used. As the Poly I:C, Poly ICLC can be used.As the TLR3 agonist, RIBOXXOL can be used.

Examples of the TLR3 agonist include Poly I:C, Poly ICLC (product name:“Hiltonol”), RIBOXXOL, Ampligen, IPH-3102, cM362-139 and cM362-140.

As the TLR3 agonist, preferably a Poly I:C is used. Of the Poly I:Cs,Poly ICLC (product name: “Hiltonol”) stabilized with Poly-Lysine andCarboxymethylcellulose, is used.

In the specification, the “TLR4 agonist” refers to a molecule whichbinds to TLR4 and provides the same stimulus as the stimulus provided bya natural ligand bound to TLR4. TLR4 recognizes a bacterium-derivedlipopolysaccharide (LPS) to activate the innate immune system. As theTLR4 agonist, MPL is known; however, the TLR4 agonist is not limited tothis. A salt of MPL may be used in the present invention; for example, asodium salt thereof can be used.

Examples of the TLR4 agonist include MPL, MPLA, OM-174 (CRX-527),Picibanil (OK-432) and ONO-4007.

In the specification, the “TLR5 agonist” refers to a molecule whichbinds to TLR5 and provides the same stimulus as the stimulus provided bya natural ligand bound to TLR5. TLR5 recognizes a bacterium-derivedFlagellin to activate the innate immune system. As the TLR5 agonist,Flagellin (proteins) derived from various bacteria or recombinantFlagellin proteins are known; however, the TLR5 agonist is not limitedto these.

Examples of the TLR5 agonist include CBLB502.

In the specification, the “TLR6 agonist” refers to a molecule whichbinds to TLR6 and provides the same stimulus as the stimulus provided bya natural ligand bound to TLR6. TLR6 recognizes, e.g., a lipoprotein andactivate the innate immune system.

Examples of the TLR6 agonist include Diacylated lipopeptides,Lipoproteins, Lipoteichoic acid and Zymosan.

In the specification, the “TLR7 agonist” refers to a molecule whichbinds to TLR7 and provides the same stimulus as the stimulus provided bya natural ligand bound to TLR7.

Also, in the specification, the “TLR8 agonist” refers to a moleculewhich binds to TLR8 and provides the same stimulus as the stimulusprovided by a natural ligand bound to TLR8.

TLR7 and TLR8 recognize virus-derived single stranded RNA to activatethe innate immune system. As the TLR7/8 agonist, Imiquimod is known;however, the TLR7/8 agonist is not limited to this. A salt of Imiquimodmay be used in the present invention; for example, a sodium salt thereofcan be used.

Examples of the TLR7/8 agonist include Imiquimod (S-26308, R-837),Resimiquimod (R-848), 3M-052, 852A, VTX-1463, VTX-2337, AZD8848(DSP-3025), ANA773 and TMX-101.

In the specification, the “TLR9 agonist” refers to a molecule whichbinds to TLR9 and provides the same stimulus as the stimulus provided bya natural ligand bound to TLR9. TLR9 recognizes bacterium- andvirus-derived CpG DNA to activate the innate immune system. As the TLR9agonist, CpG ODN is known; however, the TLR9 agonist is not limited tothis. A salt of CpG ODN (CpG Oligodeoxynucleotide) may be used in thepresent invention; for example, a sodium salt thereof can be used.

Examples of the TLR9 agonist include CpG ODN such as CpG, CpG-28,CpG-685 (GNKG168), CpG-1826, CpG-7909 (PF-3512676, Agatolimod, promune(registered trademark)), ODN1585, IMO-2125, IMO-2055 (EMD1201081),ISS1018, MGN-1703, MGN-1706, AVE0675, QAX-935, SAR-21609, SD-101 andDIMS0150.

In the specification, the “TLR10 agonist” refers to a molecule whichbinds to TLR10 and provides the same stimulus as the stimulus providedby a natural ligand bound to TLR10. TLR10 recognizes, e.g., an acylatedlipopeptide to activate the innate immune system.

Examples of the TLR10 agonist include an acylated lipopeptide.

In the specification “LAG-3 protein, a variant or derivative thereof”refers to LAG-3 protein, a functional variant or derivative thereof. TheLAG-3 protein to be used in the present invention may be derived fromany animal, for example, the protein can be derived from the same animalas the subject to which a medicine according to the present invention isto be administered. More specifically, if a medicine according to thepresent invention is administered to a human, human LAG-3 can be used.Human LAG-3 protein is a protein having an amino acid sequencerepresented by NCBI Accession No. P18627.5.

Examples of the functional variant of LAG-3 protein include, (i) anLAG-3 variant consisting of an amino acid sequence, which is the aminoacid sequence of LAG-3 having addition, substitution, or deletion of oneor several amino acids and having a function of LAG-3 protein requiredfor exerting the effects of the present invention, (ii) an LAG-3 varianthaving a sequence identity with the amino acid sequence of LAG-3 of atleast 80% or more, or 85% or more, 90% or more, 95% or more, 98% or moreor 99% or more and having a function of LAG-3 protein required forexerting the effects of the present invention, and (iii) a partialpolypeptide of LAG-3 protein, a variant defined in the above (i) or avariant defined in the above (ii) having a function of LAG-3 proteinrequired for exerting the effects of the present invention.

In the specification, the “one or several amino acids” refers to 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In the specification, the “amino acid” is used in the broadest sensethereof and includes natural amino acids and artificial amino acidvariants and derivatives. In the specification, examples of the aminoacid include natural protein L-amino acids; D-amino acids; chemicallymodified amino acids such as amino acid variants and derivatives;natural non-protein amino acids such as norleucine, β-alanine andornithine; and chemically synthesized compounds having amino acidcharacteristics known in the art. Examples of non-natural amino acidsinclude α-methyl amino acid (e.g., α-methylalanine), D-amino acid,histidine-like amino acid (e.g., β-hydroxy-histidine, homohistidine,α-fluoromethyl-histidine and α-methyl-histidine), amino acids with extramethylene in a side chain (“homo” amino acids) and amino acids (e.g.,cysteine acid) in which the carboxylic acid functional group amino acidin a side chain is substituted with a sulfonic acid group.

Examples of the functional derivative of LAG-3 protein include a fusionprotein of all or part of LAG-3 protein and another protein or apolypeptide, and LAG-3 protein attached with a sugar chain and/or alipid. As an example of the functional derivative of LAG-3 protein, afusion protein of LAG-3 and IgG (LAG-3Ig). Examples of the functionalderivative of LAG-3 protein include the derivatives described in Journalof Translational Medicine 2014, 12: 97.

As the functional derivative of LAG-3 protein, for example, a fusionprotein of LAG-3 and IgG, i.e., IMP321 (LAG-3Ig), can be preferablyused.

A medicine according to the present invention may contain a TLR agonistand LAG-3 protein, a variant or derivative thereof. As the TLR agonist,a TLR3 agonist or a TLR9 agonist can be preferably used and a TLR3agonist can be more preferably used. In a medicine according to thepresent invention, human LAG-3 and LAG-3Ig can be preferably used as theLAG-3 protein, a variant or derivative thereof.

As the combination of a TLR agonist and LAG-3 protein, a variant orderivative thereof, a combination of a TLR3 agonist or a TLR9 agonist,and LAG-3 protein, a variant or derivative thereof or a combination of aTLR agonist and LAG-3 protein or a derivative of LAG-3 protein ispreferable; a combination of a TLR3 agonist or a TLR9 agonist and LAG-3protein or a derivative of LAG-3 protein is more preferable; acombination of a TLR3 agonist and LAG-3 protein or a derivative of LAG-3protein is further preferable; and a combination of a TLR3 agonist and aderivative of LAG-3 protein is further more preferable.

In the combination of a TLR agonist and LAG-3 protein, a variant orderivative thereof, Poly I:C or a salt thereof can be used as the TLR3agonist, and Poly ICLC may be used as the Poly I:C. As the TLR3 agonist,e.g., Poly I:C, Poly ICLC (product name: “Hiltonol”), RIBOXXOL,Ampligen, IPH-3102, cM362-139 and cM362-140 may be used. As the TLR3agonist, Poly ICLC (product name: “Hiltonol”) can be preferably used.

In the combination of a TLR agonist and LAG-3 protein, a variant orderivative thereof, CpG ODN or a salt thereof may be used as a TLR9agonist and a sodium salt of CpG ODN may be used. As the TLR9 agonist,e.g., CpG, CpG-28, CpG-685 (GNKG168), CpG-1826, CpG-7909 (PF-3512676,Agatolimod, promune (registered trade mark)), ODN1585, IMO-2125,IMO-2055 (EMD1201081), ISS1018, MGN-1703, MGN-1706, AVE0675, QAX-935,SAR-21609, SD-101 and DIMS0150, may be used.

In the combination of a TLR agonist and LAG-3 protein, a variant orderivative thereof, as the LAG-3 protein, a variant or derivativethereof, the LAG-3 protein as mentioned above and a functional mutant ora functional derivative thereof can be used; a functional derivative ofLAG-3 protein may be used; and a human LAG-3 and a fusion protein ofLAG-3 and IgG (LAG-3Ig) may be used.

A medicine according to the present invention preferably contains a TLRagonist and LAG-3 protein, a variant or derivative thereof and furthercontains a substance for inducing a specific immune response to a cancercell. Also, in the case of a medicine further comprising a substance forinducing a specific immune response to a cancer cell, the aforementionedcombination of a TLR agonist and LAG-3 protein, a variant or derivativethereof can be used.

A medicine according to the present invention preferably contains a TLR3agonist or a TLR9 agonist, LAG-3 protein or LAG-3 protein derivative,and a substance for inducing a specific immune response to a cancercell. More preferably, a medicine according to the present inventioncontains a TLR3 agonist, LAG-3 protein or a derivative of LAG-3 protein,and a substance for inducing a specific immune response to a cancercell. Further preferably, a medicine according to the present inventioncontains a TLR3 agonist, a derivative of LAG-3 protein, and a substancefor inducing a specific immune response to a cancer cell.

If a medicine according to the present invention further contains asubstance for inducing a specific immune response to a cancer cell, acancer-antigen protein, a cancer-antigen derived peptide, nucleic acidsencoding them, a cancer antigen-presenting cell and a tumor cell may beused as the substance for inducing a specific immune response to acancer cell, and a cancer-antigen derived peptide is preferably used.

A medicine according to the present invention can be used for cancerimmunotherapy by the following mechanism. The medicine, which ispreferably administered by a subcutaneous or intradermal route, bringsabout the phenomena: (1) antigen presentation in the near dendriticcells, (2) activation of T cell priming in the near lymph node and (3)recognition and damage of a tumor at a tumor site by antigenpeptide-specific CTLs induced.

Taking a case where a TLR3 agonist is used as the TLR agonist in themedicine according to the present invention, as an example, a substancefor inducing a specific immune response to a cancer cell administeredtogether with the TLR3 agonist is presented as an antigen and T cellpriming is promoted.

Taking a case where LAG-3Ig is used as the LAG-3 protein, a variant orderivative thereof in the medicine according to the present invention,as an example, it is expected that antigen presentation to a substancefor inducing a specific immune response to a cancer cell is promoted; atthe same time, at a tumor site, an inhibitory signal against activatedCTLs by, e.g., a tumor or a macrophage expressing a MHC class IImolecule is blocked to suppress exhaustion of CTL.

It is considered that damage on a tumor is accelerated by the functionsof the TLR3 agonist and LAG-3Ig independently of a substance forinducing a specific immune response to a cancer cell simultaneouslyadministered.

In the specification, the “substance for inducing a specific immuneresponse to a cancer cell” is not particularly limited as long as it isa substance capable of inducing an immune response necessary forbreaking cancer cells in-vivo and inducing apoptosis of cancer cells;for example, a cancer-antigen protein, a cancer-antigen derived peptide,nucleic acids encoding them, a cancer antigen-presenting cell and atumor cell itself are mentioned.

In the specification, the “cancer-antigen protein” is a proteinspecially expressed on a cancer cell and recognized/attacked by theimmune system as a foreign body. The “cancer-antigen protein” may beexpressed on cancer cells of all types and a specific type of cancer. Asthe cancer-antigen protein, a protein having strong immunogenicity andnever be expressed in normal cells, is preferable. Examples of thecancer-antigen protein include, but are not particularly limited to,those described in Clin Cancer Res 2009; 15 (17) 5323. Specific examplesthereof include, but are not particularly limited to, WT1, LMP2, HPVE6,HPVE7, EGFRv III, HER-2/neu, MAGE-A3, p53nonmutant, HSP70, GPC3, MUC1,Casp8, CDAM1, cMyb, EVC1, EVC2, Helios, Fas, NY-ESO-1, PSMA, GD2, CEA,MelanA/MART1, Ras mutant, gp100, p53 mutant, Proteinase3 (PR1), bcr-abl,Tyrosinase, Survivin, PSA, hTERT, Sarcoma translocation breakpoints,EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, Androgen receptor,Cyclin B1, Polysialic acid, MYCN, PhoC, TRP-2, GD3, Fucosyl GM1,Mesothelin, PSCA, MAGE A1, sLe, CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH,ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAXS,OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3,Legumain, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR, MAD-CT-2 andFos-related antigen 1.

Suitable examples of the cancer-antigen protein include, but are notparticularly limited to, HSP70, GPC3, MUC1 and gp100.

In the specification, the “cancer-antigen derived peptide” refers to apeptide having a part of the amino acid sequence of a cancer-antigenprotein, or a peptide having a sequence, which is the same amino acidsequence as above and having addition, substitution or deletion of 1 or2 amino acids, or a peptide having a sequence identity with the aboveamino acid sequence of 90% or more, 95% or more, 98% or more, or 99% ormore and inducing immune which attacks cancer cells. The cancer-antigenderived peptide may consist of amino acid residues of 8 or more and 11or less. Such a peptide, if it is subcutaneously administered to apatient, is incorporated into antigen-presenting cells, such asdendritic cells and macrophages, and presented on the surface of thecells together with HLA molecules. Precursor cells of cytotoxic T cell(CTL) having a reactivity to the peptide presented are clonallyproliferated and mature CTLs that proliferated and differentiatedmigrate through the lymph flow into a cancer tissue. The mature CTLsattack a cancer cell on which a peptide having the same sequence as thepeptide administered is expressed to induce apoptosis.

Examples of the HSP70-derived peptide as the cancer-antigen derivedpeptide include, but are not particularly limited to, peptides describedin International Publication No. WO2016/056596. More specifically, apeptide comprising continuous 8 or more amino acid residues in the aminoacid sequence represented by any one of SEQ. ID Nos: 1 to 15 andconsisting of 11 or less amino acid residues, is mentioned.

In the present invention, if an HSP70-derived peptide is used, althoughit is not particularly limited, an HSP70-derived peptide having theamino acid sequence represented by any one of SEQ. ID Nos: 1 to 15 canbe preferably used, and an HSP70-derived peptide having the amino acidsequence represented by the SEQ. ID No: 7 is more preferably used.

Examples of the GPC3-derived peptide as the cancer-antigen derivedpeptide include, but are not particularly limited to, peptides describedin International Publication No. WO2016/143816. More specifically, apeptide comprising continuous 8 or more amino acid residues in the aminoacid sequence represented by any one of SEQ. ID Nos: 16 to 26 andconsisting of 11 or less amino acid residues, is mentioned.

In the present invention, if a GPC3-derived peptide is used, although itis not particularly limited, a GPC3-derived peptide having the aminoacid sequence represented by any one of SEQ. ID Nos: 16 to 26 can bepreferably used, a GPC3-derived peptide having the amino acid sequencerepresented by the SEQ. ID No: 16 is more preferably used.

Examples of the MUC1 derived peptide as the cancer-antigen derivedpeptide, include, but are not particularly limited to, peptidesdescribed in International Publication No. WO2016/143814. Morespecifically, a peptide comprising continuous 8 or more amino acidresidues in the amino acid sequence represented by any one of SEQ. IDNos: 27 to 39 and consisting of 11 or less amino acid residues, ismentioned.

These antigen-derived peptides are disclosed just as examples. The“substance for inducing a specific immune response to a cancer cell”used in the medicine according to the present invention is not limitedto these antigen-derived peptides.

In the specification, the “nucleic acid” is not particularly limited aslong as it is a nucleic acid encoding a cancer-antigen protein or acancer-antigen derived peptide, and includes RNA, DNA, PNA, LNA or achimeras of two or more of these. These nucleic acids can be insertedinto, e.g., a vector, in accordance with a known method, and thenadministered to patients and express a cancer-antigen protein or acancer-antigen derived peptide, in vivo.

In the specification, the “cancer antigen-presenting cell” refers to anantigen-presenting cell presenting a cancer-antigen derived peptide onthe surface thereof via binding to an HLA molecule. As theantigen-presenting cell, dendritic cells and macrophages can be used.The dendritic cells have particularly high CTL inducibility. Theantigen-presenting dendritic cells can be obtained, for example, byseparating mononuclear cells from the patient's own peripheral blood,differentiating the mononuclear cells into immature dendritic cells andthereafter further differentiating mature dendritic cells by adding acancer-antigen protein or a cancer-antigen derived peptide to a medium.

As a cancer vaccine presently under development, vaccines usingdendritic cells sensitized with a tumor cell extract, dendritic cellssensitized with a cancer antigen/GM-CSF fusion protein and a combinationof noninfectious virus-like particles of a HPV-derived L1 protein and anadjuvant, are known. These are included in the “substance for inducing aspecific immune response to a cancer cell”.

A medicine according to the present invention can contain at least one“substance for inducing a specific immune response to a cancer cell”;and may contain at least two types of “substances for inducing aspecific immune response to a cancer cells”. For example, a medicineaccording to the present invention may contain, as “the substance forinducing a specific immune response to a cancer cell”, a singlecancer-antigen derived peptide, which is derived from a single type ofcancer-antigen protein or may contain cancer-antigen derived peptides,which are derived from at least two types of cancer-antigen proteins.

A medicine according to the present invention preferably contains atleast one cancer-antigen derived peptide.

As the at least one type of cancer-antigen derived peptide, which is notparticularly limited, a single type of cancer-antigen derived peptidemay be used or at least two types of cancer-antigen derived peptides maybe used.

As the cancer-antigen protein in the cancer-antigen derived peptide,which is derived from a cancer-antigen protein, a protein selected fromthe cancer-antigen proteins listed in the “cancer-antigen protein”, canbe mentioned.

As the at least two types of cancer-antigen derived peptides, at leasttwo types of cancer-antigen derived peptides derived from differentcancer-antigen proteins may be used, or at least two types of differentcancer-antigen derived peptides derived from the same cancer-antigenprotein, may be used.

As the at least two types of different cancer-antigen derived peptides,at least two types of different proteins selected from thecancer-antigen proteins listed in the “cancer-antigen protein” can bementioned.

More specifically, as the cancer-antigen derived peptide, HSP70, GPC3,MUC1 or gp100-derived peptide is preferably used and an HSP70-derivedpeptide or a GPC3-derived peptide is more preferably used.

If a medicine according to the present invention contains a single typeof cancer-antigen derived peptide, it is preferable to use anHSP70-derived peptide or a GPC3-derived peptide.

If a medicine according to the present invention contains at least twotypes of cancer-antigen derived peptides, it is preferable to use anHSP70, GPC3, MUC1 or gp100-derived peptide as one of the at least twotypes of cancer-antigen derived peptides.

As the two types of cancer-antigen derived peptides, a combination of anHSP70-derived peptide and a cancer-antigen derived peptide except anHSP70-derived peptide, a combination of a GPC3-derived peptide and acancer-antigen derived peptide except a GPC3-derived peptide, and acombination of an HSP70-derived peptide and a GPC3-derived peptide, arepreferably used.

As the cancer-antigen derived peptide except an HSP70-derived peptideand the cancer-antigen derived peptide except a GPC3-derived peptide,cancer-antigen derived peptides, which are derived from antigenproteins, which are selected from the cancer-antigen proteins listed inthe “cancer-antigen protein” and, from which HSP70 and GPC3 arerespectively removed, are mentioned.

In the present invention, if two types of cancer-antigen derivedpeptides are used, it is preferable to use a combination of two types ofpeptides, i.e., an HSP70-derived peptide and a GPC3-derived peptide.

If a single type of cancer-antigen derived peptide is used, it ispreferable to use an HSP70, GPC3, MUC1 or gp100-derived peptide and morepreferable to use an HSP70-derived peptide or a GPC3-derived peptide.

At least two types of “substances for inducing a specific immuneresponse to a cancer cell” may be at least two types of substancesselected from cancer-antigen proteins, cancer-antigen derived peptides,nucleic acids encoding them, cancer antigen presenting cells and tumorcells. Taking a case comprising two types of substances for inducing aspecific immune response to a cancer cell, as an example, a single typeof cancer-antigen derived peptide and another type of cancer-antigenderived peptide may be contained; or a single type of cancer-antigenderived peptide and a single type of substance selected from acancer-antigen protein, a nucleic acid encoding a cancer-antigen proteinor a cancer-antigen derived peptide, a cancer antigen presenting celland a tumor cell, may be contained.

A medicine according to the present invention can be used as a cancervaccine therapy. In this case, a TLR agonist and LAG-3 protein, avariant or derivative thereof serve as adjuvants and enhance inductionof an immune response by a “substance for inducing a specific immuneresponse to a cancer cell”. As shown in Examples (later described), whenthe TLR agonist and LAG-3 protein, a variant or a derivative thereof areused in combination, an extremely high antitumor effect can be obtainedeven at a low dose at which no effect is obtained when they are usedalone.

In the specification, the “adjuvant” refers to a molecule(s), which isadministered together with a substance for inducing an immune responseto enhance induction of the immune response.

A cancer vaccine therapy can be used for prevention or treatment ofcancer. In the specification, prevention or treatment of cancer refersto causing at least one of phenomenon: a decrease in tumor size, delayor termination of increase in tumor size, inhibition (delay ortermination) of cancer metastasis, inhibition (delay or termination) ofproliferation of cancer cells, inhibition (delay or termination) ofcancer recurrence, and mitigation of one or more of symptoms associatedwith cancer.

In the specification, the term “cancer” is used in the broadest sensethereof. Examples of the cancer include, but are not limited to,astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma,ependymoma, neurilemmoma, neurofibrosarcoma, neuroblastoma, pituitarytumor (for example, for pituitary adenoma), medulloblastoma, melanoma,brain tumor, prostate cancer, head and neck cancer, esophageal cancer,renal cancer, renal cell carcinoma, pancreatic cancer, breast cancer,lung cancer, colon cancer, colorectal cancer, stomach cancer, skincancer, ovarian cancer, bladder cancer, fibrosarcoma, squamous cellcarcinoma, neuroectodermal tumor, thyroid tumor, lymphoma, leukemia,multiple myeloma, hepatocellular carcinoma, mesothelioma and epidermoidcarcinoma.

The present invention includes an adjuvant including a TLR agonist andLAG-3 protein, a variant or derivative thereof, for use in a cancervaccine therapy. The TLR agonist is an adjuvant to be administered incombination with LAG-3 protein, a variant or derivative thereof; whereasLAG-3 protein, a variant or derivative thereof can be an adjuvant to beadministered in combination with the TLR agonist. Such adjuvants may beadministered to a patient together with various “substances inducing aspecific immune response to a cancer cell”. The term “together” usedherein does not mean concurrent administration but means that a TLRagonist and LAG-3 protein, a variant or derivative thereof areadministered to a patient such that the TLR agonist and LAG-3 protein, avariant or derivative thereof can function as adjuvants in accordancewith a cancer vaccine therapy, in vivo (of a patient's body) and exvivo.

In the present invention, a medicine comprising a TLR agonist forcombined administration with LAG-3 protein, a variant or derivativethereof may be provided; or a medicine comprising LAG-3 protein, avariant or derivative thereof for combined administration with a TLRagonist may be provided.

Also, in the present invention, a medicine comprising a TLR agonist forcombined administration with a substance for inducing a specific immuneresponse to a cancer cell and LAG-3 protein, a variant or derivativethereof may be provided. The medicine comprising a TLR agonist can act,with LAG-3 protein, a variant or derivative thereof as adjuvants, toenhance induction of immune response by a substance for inducing aspecific immune response to a cancer cell, preferably, a cancer-antigenderived peptide. In the present invention, a medicine comprising LAG-3protein, a variant or derivative thereof for combined administrationwith a substance for inducing a specific immune response to a cancercell and a TLR agonist may be provided. The medicine comprising LAG-3protein, a variant or derivative thereof can act, with a TLR agonist asadjuvants, to enhance induction of immune response by a substance forinducing a specific immune response to a cancer cell, preferably acancer-antigen derived peptide.

In a medicine comprising a TLR agonist and LAG-3 protein, a variant orderivative thereof according to the present invention, the TLR agonistand LAG-3 protein, a variant or derivative thereof can be used as activeingredients of the medicine. The medicine comprising a TLR agonist andLAG-3 protein, a variant or derivative thereof as active ingredientsaccording to the present invention, as shown in Examples (describedlater), can be used as an anti-cancer agent having a high antitumoreffect. In the specification, the “anti-cancer agent” refers to an agentthat can be used for preventing or treating cancer. The medicine servingas an anti-cancer agent may contain a TLR agonist and LAG-3 protein, avariant or derivative thereof as active ingredients, and may furthercontain a substance for inducing a specific immune response to a cancercell.

In the present invention, a medicine serving as an anti-cancer agentcontains a substance for inducing a specific immune response to a cancercell, preferably, a cancer-antigen derived peptide, as an activeingredient, and may contain a TLR agonist and LAG-3 protein, a variantor derivative thereof as adjuvants.

Components of a medicine according to the present invention aredissolved in a water-soluble solvent to prepare pharmaceuticallyacceptable salts and a preparation containing the components in the saltform can be administered to patients. Examples of such pharmaceuticallyacceptable salts include physiologically acceptable water-soluble saltssuch as a sodium salt, a potassium salt, a magnesium salt and a calciumsalt, which are adjusted to have physiological pH by a buffer. Otherthan water-soluble solvents, a nonaqueous solvent can be used. Examplesof the nonaqueous solvent include alcohols such as ethanol and propyleneglycol.

A medicine according to the present invention can be used as apharmaceutical composition, which has any dosage form orally orparenterally administrated. The dosage form is not particularly limited.Examples of the dosage form of a pharmaceutical composition can includea liquid (for example, an injection), a dispersant, a suspension, atablet, a pill, a powdered drug, a suppository, a powder, a fine grain,a granule, a capsule, syrup, a nasal drop and an ear drop.

A medicine according to the present invention, if it is used as a cancervaccine, can be orally or parenterally administered. As parenteraladministration, for example, intraperitoneal administration,subcutaneous administration, intradermal administration, intramuscularadministration, intravenous administration or intranasal administrationcan be employed.

A preparation of a medicine according to the present invention can beproduced in accordance with a method known in the art. In thepreparation of a medicine according to the present invention,pharmaceutically acceptable carriers and additives (such as anexcipient, a binder, a dispersant, a disintegrant, a lubricant, asolvent, a solubilizer, a coloring agent, a flavoring agent, astabilizer, an emulsifier, a suspending agent, an absorption promoter, asurfactant, a pH adjustor, a preservative and an antioxidant) may beused. Examples of the carriers and additives include pharmaceuticallyacceptable organic solvents such as water, saline, a phosphate buffer,dextrose, glycerol and ethanol, collagen, polyvinyl alcohol,polyvinylpyrrolidone, a carboxyvinyl polymer, sodiumcarboxymethylcellulose, sodium polyacrylate, sodium alginate,water-soluble dextran, sodium carboxymethyl starch, pectin, glutamicacid, aspartic acid, methylcellulose, ethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, xanthan gum, gumarabic, casein, agar, polyethylene glycol, diglycerin, glycerin,propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid,human serum albumin, mannitol, sorbitol, lactose, glucose, corn starch,microcrystalline cellulose, a surfactant, sodium bisulfite, sodiumbisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methylparaben, phenylethyl alcohol, ammonia, dithiothreitol, betamercaptoethanol, sodium carbonate, sodium borate, sodium phosphate,sodium acetate, sodium bicarbonate, sucrose, powdered sugar, sucrose,fructose, glucose, lactose, reduced malt sugar syrup, powdered reducedmalt sugar syrup, glucose fructose syrup, fructose glucose sugar, honey,erythritol, aspartame, saccharin, saccharin sodium and gelatin.

When a medicine according to the present invention contains a peptide,an absorption enhancer for improving absorption of a poorly absorbabledrug (hard-to-transmucosally absorbed drug), such as a peptide, may beused. Examples thereof include surfactants such as a polyoxyethylenelauryl ether, sodium lauryl sulfate and saponin; bile salts such asglycocholic acid, deoxycholic acid and taurocholic acid; chelatingagents such as EDTA and salicyl bile salt; fatty acids such as caproicacid, capric acid, lauric acid, oleic acid, linoleic acid and mixedmicelle; an enamine derivative, a N-acyl collagen peptide, a N-acylamino acid, a cyclodextrin, a chitosan and a nitric oxide donor.

When a medicine according to the present invention contains a peptide,the medicine is encapsulated in e.g., polylactic acid/glycolic acid(PLGA) microcapsules or allowed to adsorb to porous hydroxyapatite fineparticles to prepare a sustained release medicine, or the medicine isapplied to a pulsed release iontophoresis patch system to prepare atransdermal absorbent.

A medicine according to the present invention may be a singlepreparation containing a TLR agonist and LAG-3 protein, a variant orderivative thereof or may be a combination of different preparations,i.e., a preparation containing a TLR agonist and a preparationcontaining LAG-3 protein, a variant or derivative thereof.

Also a medicine according to the present invention may be a singlepreparation containing a substance for inducing a specific immuneresponse to a cancer cell, a TLR agonist and LAG-3 protein, a variant orderivative thereof or may be a combination of different preparations,i.e., a preparation containing a substance for inducing a specificimmune response to a cancer cell, a preparation containing a TLRagonist, and a preparation containing LAG-3 protein, a variant orderivative thereto. More specifically, a preparation containing twotypes of components selected from a substance for inducing a specificimmune response to a cancer cell, a TLR agonist and LAG-3 protein, avariant or derivative thereof may be used in combination with apreparation containing the other remaining component. As long as acombination of three types of components is provided, a preparationcontaining any two types of components and a preparation containing anytwo types of components, may be used in combination.

A medicine according to the present invention may be provided in kitform. In the case of a kit, the kit may contain a TLR agonist and LAG-3protein, a variant or derivative thereof, or may contain a substance forinducing a specific immune response to a cancer cell, a TLR agonist andLAG-3 protein, a variant or derivative thereof.

A medicine according to the present invention, if it is used as a cancervaccine, may contain additional adjuvants. Non-limiting examples of theadditional adjuvants include sedimentary adjuvants such as aluminumhydroxide, sodium hydroxide, aluminum phosphate, calcium phosphate, alumand carboxyvinyl polymer, and oily adjuvants such as Freund's completeadjuvant, Freund's incomplete adjuvant, liquid paraffin, lanolin,montanide ISA763AV and montanide ISA51.

A medicine according to the present invention may be used in combinationwith other anti-cancer agents in any embodiment and may be used incombination with a radiation therapy and a surgical treatment. Examplesof other anti-cancer agent include low molecular compounds such asadriamycin, daunomycin, mitomycin, cisplatin, vincristine, epirubicin,methotrexate, 5-fluorouracil, aclacinomycin, nitrogen mustard,cyclophosphamide, bleomycin, daunorubicin, doxorubicin, vincristine,vinblastine, vindesine, tamoxifen, and dexamethasone; and proteins suchas cytokines activating immunocompetent cells (for example, humaninterleukin 2, human granulocyte macrophage colony stimulating factor,human macrophage colony stimulating factor, and human interleukin 12).

The present invention includes a method for treating cancer byadministering a medicine according to the present invention in atherapeutically effective dose. The therapeutically effective dose canbe appropriately determined by those skilled in the art depending upone.g., the symptoms, age, sex, body weight and sensitivity difference ofthe patient, the administration method, the administration interval andtype of preparation.

In the present invention, it is possible to treat or prevent a patient'sdisease by administering a TLR agonist and LAG-3 protein, a variant orderivative thereof to a patient in need thereof. In the presentinvention, a method for inducing a specific immune response to a cancercell by administering a TLR agonist and LAG-3 protein, a variant orderivative thereof, to a patient in need thereof, is provided.

In treating or preventing a patient's disease or in a method forinducing a specific immune response to a cancer cell, a substance forinducing a specific immune response to a cancer cell, preferably, acancer-antigen derived peptide, may be further administered.

A medicinal effect of a substance for inducing a specific immuneresponse to a cancer cell, preferably a cancer-antigen derived peptide,can be more strongly exerted by inducing a specific immune response to acancer cell.

A medicine according to the present invention is preferably used forcombined administration.

The use of a medicine according to the present invention in combinedadministration means that a TLR agonist and LAG-3 protein, a variant orderivative thereof may be administered to a patient in any combinationat the same time or different times. If a medicine according to thepresent invention contains a substance for inducing a specific immuneresponse to a cancer cell, the substance for inducing a specific immuneresponse to a cancer cell, a TLR agonist and LAG-3 protein, a variant orderivative thereof may be administered in any combination at the sametime or different times.

In administering components at the same time, the components may beadministered in single dosage form at the same time; more specifically,a TLR agonist and LAG-3 protein, a variant or derivative thereof may bemixed at the time of administration to prepare a dosing preparation andadministered at the same time.

In administering components at different times, a TLR agonist isadministered, and thereafter, LAG-3 protein, a variant or derivativethereof may be administered; or LAG-3 protein, a variant or derivativethereof is administered and thereafter a TLR agonist may beadministered.

In administering components at the same time if a medicine according tothe present invention contains a substance for inducing a specificimmune response to a cancer cell, the components may be administered insingle dosage form at the same time; more specifically, the substancefor inducing a specific immune response to a cancer cell, a TLR agonistand LAG-3 protein, a variant or derivative thereof may be mixed at thetime of administration to prepare a dosing preparation and administeredat the same time.

In administering components at different times, the substance forinducing a specific immune response to a cancer cell is administered,and thereafter, a TLR agonist and LAG-3 protein, a variant or derivativethereof are administered at the same time or different times; or the TLRagonist and LAG-3 protein, a variant or derivative thereof may beadministered at the same time or different times, and thereafter, thesubstance for inducing a specific immune response to a cancer cell maybe administered. Alternatively, one of the TLR agonist and LAG-3protein, a variant or derivative thereof is administered, andthereafter, the substance for inducing a specific immune response to acancer cell is administered, and then, the other one of the TLR agonistand the LAG-3 protein, a variant or derivative thereof, may beadministered. In administering the components at different times, thecomponents may be administered based on the properties and the doseinterval of individual components, in other words, based on the dosingregimens of the individual components.

The disclosures of all Patent Literatures and Non Patent Literaturescited in the specification are incorporated herein in their entirety byreference.

EXAMPLES

Now, the present invention will be described based on Example; however,the present invention is not limited to this. The present invention canbe modified in various ways by those skilled in the art withoutdeparting from the scope of the present invention and such modificationis included in the scope of the present invention.

1. Cancer Vaccine

In accordance with the protocol shown in FIG. 1 , the effects ofadjuvants in cancer vaccines using a cancer-antigen derived peptide werecompared.

Material

As cancer model mice, DBA/2 mice, to which P815 cells(DBA/2mouse-derived mouse mastocytoma) were grafted, were put in use.

As a cancer-antigen derived peptide, a peptide (hereinafter referred toas “PIA peptide”) consisting of a partial sequence of PIA protein wasused, which is a tumor antigen of P815 tumor and known to berestrictively presented by an MHC H-2Ld. The amino acid sequence of PIApeptide are represented by LPYLGWLVF (SEQ. ID No: 40).

As PIA CTL, a T cell expressing a T cell receptor and recognizing PIApeptide was used. In the experiment, the spleen was taken out from aPIA-CTL transgenic mouse that the present inventors possessed and apositive ratio was checked by TCR Vα 8.3 (TCR marker obtained by geneintroduction). Based on the positive ratio, the number of PIA-CTL cellswas determined and administered.

As the adjuvants, the following substances were used.

Whole pertussis bacterial cell (PT) (BioFarma, Bandung, Indonesia)

Poly I:C (TLR3 agonist) (Invivogen, SanDiego, USA)

CpG (TLR9 agonist) (Invivogen, SanDiego, USA)

IFA (incomplete Freund's adjuvant) (Seppic, Paris, France)

LAG-3Ig (Adipogen, SanDiego, USA)

Method

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in anamount of 5×10⁵ cells per mouse. The day of inoculation was specified asDay 0. On Day 8, PIA CTL (2.5×10⁵ cells per mouse) were intravenouslyinjected. On Day 9 and Day 16, an admixture of PIA peptide (50 μg permouse) and an adjuvant in PBS was subcutaneously injected. Mice weredivided into 9 groups each consisting of 5 mice and the followingadjuvants were used, respectively.

Group 1: PBS (Control)

Group 2: IFA (50 μL/mouse)

Group 3: PT (1×10⁸/mouse)

Group 4: Poly I:C (50 μg/mouse)

Group 5: Poly I:C (50 μg/mouse)+CpG (10 μg/mouse)

Group 6: LAG-3Ig (1 μg/mouse)

Group 7: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Results

Changes in tumor size (mm³) of all mice on and after Day 7 are shown inFIGS. 2 to 8 .

In all of Groups 1 to 6, tumor sizes gradually increased and mice mostlydied in the middle. The number of mice survived until Day 70 withoutincreasing tumor size was 0 in Groups 2, 3 and 6, 1 in Groups 1 and 4,and 3 in Group 5; however, in Group 7, an increase in tumor size was notobserved in all 5 mice and the mice survived until Day 115.

2. Infiltration of Immune Cells into Cancer Tissue

Cancer model mice were prepared by subcutaneously inoculating P815 tumorcells (5×10⁵ cells per mouse) to DBA/2 mice. The day of inoculation wasspecified as Day 0. On Day 8, PIA CTL (2.5×10⁵ cells per mouse) wereintravenously injected. Day 9 and Day 14, an admixture of PIA peptide(50 μg per mouse) and an adjuvant in PBS was subcutaneously injected.The following adjuvants were used in respective mouse groups.

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse)

Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

On Day 21, a tumor tissue was taken and slides of tumor tissue sectionswere prepared. Thereafter, a tissue image was observed by staining withhematoxylin-eosin; at the same time, cell nuclei and immune cells (CD4cells and CD8 cells) were stained with a fluorescent dye.

The following reagents were used in the fluorescent tissue staining.

Cell nucleus: ProLongR Gold Antifade Reagent with DAPI (Invitrogen)

CD4 cell primary antibody: Rat Anti-Mouse CD4 Purified IgG2b. Clone:GK1.5 (eBioscience)

CD4 cell secondary antibody: Mouse monoclonal (2B 10A8) Anti-Rat IgG2bheavy chain (Alexa FluorR 647), (abcam)

CD8 cell primary antibody: Rat Anti-Mouse CD8a Purified IgG2a. Clone:53-6.7 (eBioscience)

CD8 cell secondary antibody: Mouse monoclonal (2A 8F4) Anti-Rat IgG2aheavy chain (Alexa FluorR 488) (abcam)

Tissue images stained with hematoxylin-eosin are shown in FIG. 9A andimages of cell nucleus and immune cells stained with a fluorescent dyeare shown in FIG. 9B. When LAG-3Ig and poly I:C were used in combinationas an adjuvant, significant infiltration of CD4 cells and CD8 cells intoa tumor tissue was observed.

3. Maintenance of Ability to Reject the Same Tumor in Mice Once Rejecteda Tumor

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in anamount of 5×10⁵ cells per mouse and used as cancer model mice. The dayof inoculation was specified as Day 0. On Day 8, P1A CTL (2.5×10⁵ cellsper mouse) were intravenously injected. On Day 9 and Day 14, anadmixture of P1A peptide (50 μg per mouse) and an adjuvant in PBS wassubcutaneously injected. As an adjuvant, LAG-3Ig (1 μg/mouse)+Poly I:C(50 μg/mouse) was used.

Subsequently, to DBA/2 mice caused no increase in tumor size even ifP815 tumor cells were inoculated, and determined as rejecting a tumor,and naive DBA/2 mice to which no treatment was applied, again P815 tumorcells (1×10⁶ cells per mouse) or L1210 tumor cells (1×10⁶ cells permouse) were subcutaneously inoculated. To DBA/2 mice determined asrejecting a tumor, the tumor cells were again inoculated on Day 115after the first inoculation of P815 tumor cells.

FIG. 10 (left) shows changes in average tumor size (mm³) of mice afterinoculation of L1210 cells; whereas, FIG. 10 (right) shows changes inaverage tumor size (mm³) of mice after inoculation of P815 cells. Inboth figures, open circle represents DBA/2 mice determined as a tumorrejection mice, and closed circle represents naive DBA/2 mice.

In naive DBA/2 mice, an increase in tumor size was observed even ifeither one of P815 cells and L1210 cells were inoculated. In contrast,in mice determined as rejecting a tumor growth of P815 cells, anincrease in tumor size by inoculation of another type of tumor cells,i.e., L1210 cells, was observed; however, an increase in tumor size byP815 cell inoculation was not observed. It was confirmed that an abilityto reject the same tumor is maintained.

4. Determination of the Proliferative Ability of Immune Cells andMeasurement of the Cytokines in Cell Supernatant

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in anamount of 5×10⁵ cells per mouse to prepare cancer model mice. The day ofinoculation was specified as Day 0. On Day 8, PIA CTL (2.5×10⁵ cells permouse) were intravenously injected. On Day 9 and Day 14, an admixture ofPIA peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneouslyinjected. The following adjuvants were used in respective mice groups.

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse)

Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Of the lymph nodes in the axilla and inguinal region, the lymph nodeclose to a tumor site was taken on Day 21 and immune cells wereseparated. The immune cells (1.5×10⁵ cells) and P815 cells (4×10⁴ cells)irradiated with 100 Gy were co-cultured for 3 days.

Immune cell proliferative ability was determined by adding ³H-thymidine(37 KBq/well) to the culture supernatant and measuring the radioactivityof ³H-thymidine taken in the cells, 4 hours later.

The amounts of cytokines in the cell supernatant were measured by usingBio-Plex Pro mouse cytokine 23-Plex Immunoassay kit (BIO-RAD).

Measurement results of immune cell proliferative ability are shown inFIG. 11A and measurement results of cytokines are shown in FIG. 11B.

In a case of using a combination of LAG-3Ig and poly I:C as an adjuvant,immune cell proliferative ability increased. Among the cytokines, IFN-γ,GM-CSF, IL-4, IL-5 and IL-17A were found to increase in production whenLAG-3Ig and poly I:C were used in combination as an adjuvant.

5. Measurement of Cell-Surface Marker Molecules of Immune Cells

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in anamount of 5×10⁵ cells per mouse to prepare cancer model mice. The day ofinoculation was specified as Day 0. On Day 8, PIA CTL (2.5×10⁵ cells permouse) were intravenously injected. On Day 9 and Day 14, an admixture ofPIA peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneouslyinjected. The following adjuvants were used in respective mice groups.

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse)

Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Of the lymph nodes in the axilla and inguinal region, the lymph nodeclose to a tumor site was taken on Day 21, CD8 and Vα 8.3 expressionpositive cell group (killer T cells) or CD4 and Vα 8.3 expressionpositive cell group (helper-T cells) were collected.

Expression levels of cell-surface marker molecules (on CD4 cells and CD8cells), i.e., PD-1, BTLA, TIGIT and LAG-3, were measured. As antibodiesagainst the cell surface marker molecules, the following molecules wereused.

PD-1: Anti-mouse CD279 (PD-1) PE. Clone: J43 (eBioscience)

BTLA: Anti-mouse CD272 (BTLA) PE. Clone: 8F4 (eBioscience)

TIGIT: PE anti-mouse TIGIT (Vstm3) Antibody. Clone: 1G9 (BioLegend)

LAG-3: Anti-mouse CD223 (Lag-3) PE. Clone: eBioC9B7W (C9B7W)(eBioscience)

The results of CD8 cells are shown in FIG. 12A and the results of CD4cells are shown in FIG. 12B.

When a combination of LAG-3Ig and poly I:C was used as an adjuvant,significant decreases in expression level of PD-1, TIGIT and LAG-3 wereobserved both in CD4 positive cells and CD8 positive cells; however, thedecrease in expression level of BTLA was low.

6. Measurement of Cytokines Using B16-F10 Melanoma Inoculation ModelMice

To C57BL/6 mice, B16-F10 melanoma cells (1×10³ cells per mouse) weresubcutaneously inoculated to prepare cancer model mice. The day ofinoculation was specified as Day 0. On Day 8, an admixture of gp100peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneouslyinjected. The following adjuvants were used in respective mice groups.The amino acid sequence of gp100 peptide is represented by KVPRNQDWL(SEQ. ID No: 41).

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse) Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50μg/mouse)

Of the lymph nodes in the axilla or inguinal region, the lymph nodeclose to a tumor site (or both lymph nodes) was taken on Day 14 andimmune cells were separated. The immune cells (3×10⁵ cells per well)were cultured in the presence of gp100 peptide (10, 5, 2.5, or 0 μg/mL).

Immune cell proliferative ability was determined by adding ³H-thymidine(37 KBq/well) to the culture supernatant and measuring the radioactivityof ³H-thymidine incorporated in the cells in the final 10 hours of theculture period of 3 days.

The IFN-γ production amount in the cell supernatant obtained byculturing 3 days in the presence of gp100 peptide (10 μg/m) was measuredby Bio-Plex Pro mice cytokine 23-Plex Immunoassay kit (BIO-RAD).

Measurement results of immune cell proliferative ability are shown inFIG. 13A and measurement results of IFN-γ production amount are shown inFIG. 13B.

In FIG. 13A, open circle represents the results of Group 1, closedsquare Group 2, open square Group 3 and closed circle Group 4.

Even if B16-F10 melanoma inoculation model mice were used, if acombination of LAG-3Ig and poly I:C was used as an adjuvant, an increaseof immune cell proliferative ability specific to gp100 tumor antigen andan increase of IFN-γ production were observed.

It was confirmed that when a combination of LAG-3Ig and poly I:C wasused as an adjuvant, a significant activation effect on the immunesystem can be exerted regardless of the tumor model system and the typeof peptide serving as an immune antigen.

7. Suppression Effect of Tumor Growth by Immunoadjuvant Combination

The following substances were used as adjuvants.

Poly I:C (TLR3 agonist) (Invivogen, SanDiego, USA)

RIBOXXOL (TLR3 agonist) (Riboxx, Radebeul, Germany)

MPL (TLR4 agonist) (Invivogen, SanDiego, USA)

Imiquimod (TLR7/8 agonist) (Invivogen, SanDiego, USA)

CpG (TLR9 agonist) (Invivogen, SanDiego, USA)

LAG-3Ig (Adipogen, SanDiego, USA)

P815 tumor cells were subcutaneously inoculated to DBA/2 mice in anamount of 5×10⁵ cells per mouse to prepare cancer model mice. The day ofinoculation was specified as Day 0. On Day 7, PIA CTL (2.5×10⁵ cells permouse) were intravenously injected. Day 8 and Day 15, an admixture ofPIA peptide (50 μg per mouse) and an adjuvant in PBS was subcutaneouslyinjected. Mice were divided into 9 groups each consisting of 4 or 5 miceand the following adjuvants were used respectively.

Group 1: PIA peptide alone (control)

Group 2: Poly I:C (50 μg/mouse)

Group 3: Poly I:C (50 μg/mouse)+LAG-3Ig (1 μg/mouse)

Group 4: No PIA peptide, Poly I:C (50 μg/mouse)+LAG-3Ig (1 μg/mouse)alone

Group 5: RIBOXXOL (100 μg/mouse) Group 6: RIBOXXOL (100μg/mouse)+LAG-3Ig (1 μg/mouse)

Group 7: MPL (10 μg/mouse)+LAG-3Ig (1 μg/mouse) Group 8: Imiquimod (50μg/mouse)+LAG-3Ig (1 μg/mouse)

Group 9: CpG (10 μg/mouse)+LAG-3Ig (1 μg/mouse)

Change in tumor size (mm³) with respect to all mice on and after Day 7are shown in FIGS. 14 to 22 .

In Group 1, 5 out of 5 mice all died up to Day 40. Four out of 5 mice inGroup 2, 5 out of 5 mice in Group 3, 3 out of 5 mice in Group 6, 2 outof 5 mice in Group 4 and 5 and 1 out of 5 mice in Groups 7 to 9survived. In addition, 4 out of 5 mice in Group 3 and 3 out of 5 mice inGroup 6 survived up to Day 66. A combination effect of adjuvants wasdemonstrated by comparing between Groups 2 and 3 and Groups 5 and 6.

8. Tumor Growth Inhibitory Effect by Combination of Immuno-Adjuvants inB16-F10 Melanoma Inoculation Model Mice

To C57BL/6 mice, B16-F10 melanoma cells (1×10⁵ cells per mouse) weresubcutaneously inoculated to prepare cancer model mice. The day ofinoculation was specified as Day 0. On Day 5 and Day 12, twice in total,an admixture of gp100 peptide (50 μg per mouse) and an adjuvant in PBSwas subcutaneously injected. The following adjuvants were used inrespective mice groups.

Group 1: IFA (50 μL/mouse)

Group 2: Poly I:C (50 μg/mouse)

Group 3: LAG-3Ig (1 μg/mouse)

Group 4: LAG-3Ig (1 μg/mouse)+Poly I:C (50 μg/mouse)

Changes in tumor size (mm³) of all mice on and after Day 5 are shown inFIGS. 23 to 26 .

Five mice all died on Day 31 in Group 1, on Day 35 in Group 2 and on Day37 in Group 3. In contrast, five mice died in Group 4 on Day57. Comparedto Group 1, a significant effect of extending survival time was notobserved in Groups 2 and 3; however, the survival time of Group 4 wassignificantly extended compared to any one of Groups 1, 2 and 3.

9. Measurement of Vaccine Specific Immune Response Using Patient BloodTreated with Vaccine

To esophageal cancer patients having an HLA molecule of TypeHLA-A*02:07/24:02 and hepatocellular carcinoma patients having an HLAmolecule of Type HLA-A*24:02/26:01, a mixed solution of 2 mg of an HSP70peptide (YGAAVQAAI: SEQ. ID No: 7), 2 mg of a GPC3 peptide (MVNELFDSL:SEQ. ID No: 16), 1 mg of IMP321 (LAG-3Ig) and 1.4 mg of “Hiltonol”(product name of Poly ICLC) in saline, was divided into four portionsand each subcutaneously inoculated at 4 sites in the proximity of thelimb groin area once a week in the first 2 months and once two weeks inthe following month. After administration 10 times in total, 50 mL ofthe peripheral blood was sampled.

From the peripheral blood, PBMCs (peripheral blood mononuclear cells)were separated by using Ficoll-Paque Plus density gradient solution (GEHealthcare Bio-sciences), and the PBMCs were seeded in AIM-V+FBS culturemedium in a 24-well plate at a ratio of 1×10⁶ cells per well andcultured.

On Day 1, Day 4, Day 8 and Day 12, 100 units/mL of recombinant IL2(rIL2; Novartis) was added. On Day 0 and Day 7, 10 μg/mL of HSP70peptide or GPC3 peptide, or HIV peptide (RYLRDQQLL: SEQ. ID No: 42) as anegative control or EBV peptide (TYGPVFMCL: SEQ. ID No: 43) as apositive control were added, and culture of the PBMCs was continued. OnDay 14, CD8+ T cells were separated and recovered by negative selectionusing MACS beads.

ELISPOT assay was carried using the human IFN-γ ELISPOT PLUS kit(Mabtech). Using a 12-well plate previously coated with an anti-IFN-γantibody, CD8+T responder cells (1×10⁴ cells/well) were culturedtogether with tumor cells (2×10⁴ cells/well) as a stimulator at 37° C.for 48 hours.

After an anti-IFN-γ antibody (7-B6-1) biotinylated was added as asecondary antibody and culture was carried out for 2 hours, an HRP(Horse Raddish Peroxidase) reagent was added and further staining wascarried out by use of a TMB (tetramethylbenzidine) reagent.

The spots stained were automatically counted by use of the ImmunoSPOT S4(Cellular Technology Ltd.).

The results are shown in FIG. 27A and FIG. 27B.

After an HSP70 peptide and a GPC3 peptide were administered 10 times intotal, the number of spots associated with IFN-γ productionadvantageously increases compared to the negative control. It can beconfirmed that a vaccine-specific immune response occurs.

Further, to hepatocellular carcinoma patients having an HLA molecule ofType HLA-A*24:02/24:02, colorectal cancer patients having an HLAmolecule of Type HLA-A*02:01/11:01 and esophageal cancer patients havingan HLA molecule of Type HLA-A*02:06/26:01 before vaccination, a mixedsolution of 2 mg of an HSP70 peptide (YGAAVQAAI: SEQ. ID No: 42), 2 mgof a GPC3 peptide (MVNELFDSL: SEQ. ID No: 43), 1 mg of IMP321 (LAG-3Ig)and 1.4 mg of “Hiltonol” (product name of Poly ICLC) in saline wasdivided into four portions and each subcutaneously inoculated at 4 sitesin the proximity of the limb groin area for a month, two months and twomonths at intervals of a week (administered 4 times, 8 times and 8times) and 50 mL of the peripheral blood was sampled.

The peripheral blood samples each were separated and cultured in thesame manner as the peripheral blood samples from the above esophagealcancer patients and the hepatocellular carcinoma patients, and stainedwith the same reagent. The stained spots were automatically counted inthe same manner as above.

The results are shown in FIG. 28A, FIG. 28B and FIG. 28C.

After an HSP70 peptide and a GPC3 peptide were administered 4 times or 8times, the number of spots associated with IFN-γ productionadvantageously increases compared to the negative control. It can beconfirmed that a vaccine-specific immune response occurs.

Sequence Listing Free Text

SEQ. ID Nos: 1 to 15 represent the amino acid sequences of HSP70-derivedpeptides as a cancer-antigen derived peptide.

SEQ. ID Nos: 16 to 26 represent the amino acid sequences of GPC3-derivedpeptides as a cancer-antigen derived peptide.

SEQ. ID Nos: 27 to 39 represent the amino acid sequences of MUC1 derivedpeptides as a cancer-antigen derived peptide.

SEQ. ID No: 40 represents the amino acid sequence of PIA peptide.

SEQ. ID No: 41 represents the amino acid sequence of gp100 peptide.

SEQ. ID No: 42 represents the amino acid sequence of HIV peptide.

SEQ. ID No: 43 represents the amino acid sequence of EBV peptide.

[Sequence Listing]

The invention claimed is:
 1. A pharmaceutical composition comprising aToll-like receptor agonist, lymphocyte activation gene 3 (LAG-3) proteinor a fusion protein of an extracellular domain of LAG-3 protein and IgG,and an HSP70-derived peptide consisting of SEQ ID NO: 7 and aGPC3-derived peptide consisting of SEQ ID NO:
 16. 2. The pharmaceuticalcomposition according to claim 1, wherein the Toll-like receptor agonistis a Toll-like receptor 3 agonist or a Toll-like receptor 9 agonist. 3.The pharmaceutical composition according to claim 1, wherein theToll-like receptor agonist is Poly I:C or a salt thereof.
 4. Thepharmaceutical composition according to claim 3, wherein the Poly I:C isPoly ICLC.
 5. A pharmaceutical composition comprising a preparationcomprising a Toll-like receptor agonist, a preparation comprisinglymphocyte activation gene 3 (LAG-3A protein or a fusion protein of theextracellular domain of LAG-3 protein and IgG, and a preparationcomprising an HSP70-derived peptide consisting of SEQ ID NO: 7 and aGPC3-derived peptide consisting of SEQ ID NO: 16 wherein thepreparations are formulated for combined administration.
 6. Thepharmaceutical composition according to claim 5, wherein the Toll-likereceptor agonist is a Toll-like receptor 3 agonist or a Toll-likereceptor 9 agonist.
 7. The pharmaceutical composition according to claim5, wherein the Toll-like receptor agonist is Poly I:C or a salt thereof.8. The pharmaceutical composition according to claim 7, wherein the PolyI:C is Poly ICLC.